This invention provides a method for the combination of human relaxin A- and B-chains or human relaxin A- and B-chain analogs to produce useful yields of biologically active human relaxin or human relaxin analog. In particular the invention comprises combining the reduced human relaxin A- and B-chains or analogs thereof under conditions which include a pH greater than about 7.0 and which are mildly denaturing with respect to the human relaxin B-chain. These conditions provide a milieu for formation of biologically active human relaxin or an analog thereof by maintaining the mixture at a temperature of from about 15.degree. C. to 30.degree. C. with gradual exposure to air oxygen over the course of the reaction. This invention also provides biologically active analogs of human relaxin. This invention further provides a method of effecting parturition using human relaxin or an analog thereof as the sole active agent.
Human relaxin is an ovarian peptide responsible for remodelling the reproductive tract before parturition, thus facilitating the birth process. Hisaw, F. L., Pros. Soc. Exp. Bio. Med. 23, 661-663 (1926); Schwabe, C. et al. Biochem. Biophy. Res. Comm. 75, 503-570 (1977); James, R. et al., Nature 267, 544-546 (1977). While predominantly a hormone of pregnancy, relaxin has also been detected in the non-pregnant female as well as in the male. Bryant-Greenwood, G. D., Endocrine Reviews 3, 62-90 (1982) and Weiss, G., Ann. Rev. Physio. 46, 43-52 (1984).
The amino acid sequences of relaxim from pig, rat, tiger shark, dogfish shark and human have been established. The hormone consists of two peptide chains, referred to as A and B, joined by disulfide bonds with an intro-chain disulfide loop in the A-chain in a manner analogous to that of insulin. However, a surprising and important difference between relaxin and most other peptide hormones, including insulin, is the considerable structural variation between species. For example, pig, rat and human relaxins differ in over 50% of amino acid positions. These differences explain the poor immunological cross-reactivity between relaxins of different species and also a number of the observed differences in their specific biological activity.
The application of recombinant DNA technology has led to the isolation and characterization of the genes coding for human relaxins. Hudson, P. et al., Nature 301, 628-631 (1983) and Hudson, P. et al., The EMBO Journal 3, 2333-2339 (1984). Analysis of the nucleotide sequence from cDNA and genomic clones reveals the structural organization of human relaxin to include a signal peptide of 25 residues, followed by a B-chain of about 32 to 33 amino acids, a C-peptide of about 105 amino acids, and an A-chain of 24 amino acids. In the case of human relaxin an intron interrupts the coding region of the C-peptide. The physiological role of the C-peptide, which is considerably longer than the C-peptide of insulin, and the nature of the enzymes responsible for removal of the C-peptide from the ends of the A- and B-chain are unresolved issues.
In the case of human relaxin two separate gene sequences have been identified. Id. Only one of these genes (H2) is expressed in the ovary during pregnancy, and it is unclear whether the other gene is expressed at another tissue site, or whether it represents a pseudo-gene. The two human relaxin genes show considerable nucleotide and amino acid homology to each other, particularly in the B and C peptide. However, there are some notable regions of sequence divergence, particularly in the amino terminal region of both A- and B-chains. See FIG. 1. The fact that H2 relaxin is synthesized and expressed in the ovary suggests that this is the sequence which is involved in the physiology of pregnancy. In a recent paper Johnston, P. D. et al., In Peptides: Structure and Function, Proc. Ninth American Peptide Symposium, Deber, C. M., Hruby, V. I. and Kopple, F. D. (eds.) (Pierce Chem Co., 1985) tested synthetic human relaxin (H2) and certain human relaxin analogs for biological activity. They defined a relaxin core necessary for biological activity as well as certain amino acid substitutions for methionine which did not affect biological activity. Id.
FIG. 1 compares the known amino acid sequences of relaxins from different species. In addition to the six cysteine residues and flanking glycine residues, only the isoleucine at position 7 in the B-chain, the arginine at positions 12 and 16, and the leucine at position 32 have been conserved. The cysteine residues are clearly essential to maintaining the overall disulfide bond configuration. Blundell, T. et al. In: Bigazzi, M., Greenwood, F. C., Gaspari, F. (eds.) Biology of Relaxin and its Role in the Human, (Excerpta Medica, Amsterdam, 1983) pp. 14-21. The species variation in the amino acid sequence at most positions in the relaxin molecule, i.e. in the A- and B-chains which comprise the active portion of the relaxin protein, is remarkable. This is in marked contrast to the situation with virtually all other peptide hormone families including insulin. Another feature of the relaxin structure is the variation in length seen at the amino and carboxyl terminal regions of the B-chain, and to a lesser extent at the amino terminus of the A-chain.
The chemical synthesis of relaxin has been particularly difficult largely as a result of the unusual solubility and structural characteristics of the isolated B-chain. Tregear, G. W. et al., In: Bigazzi, M., Greenwood, F. C. and Gaspari, F. (eds.), Biology of Relaxin and its Role in the Human, (Excerpta Medica, Amsterdam, 1983), pp. 42-55.
As discussed above human relaxin and in fact mammalian relaxin generally has some structural similarity to insulin. Both insulin and relaxin have inter-and intra-chain disulfide bonds between the two chains. James, R. et al. Nature 267; 544 (1977) and Schwabe, C. and McDonald, J. R. Science 197, 914 (1977). It was presumed that synthetic strategies which were used successfully for insulin, Busse, W. D. and Carpenter, F. J., Biochemistry 15, 1649 (1976), Katsoyannis, P. G. et al., Biochemistry 6, 2656 (1967) and Kung, Y. T. et al., Scientia Sinica 15, 544 (1966), would also be applicable to relaxin. Tregear, G. et al. In: Relaxin, G. D. Bryant-Greenwood, Niall, H. D. and Greenwood, F. C. (eds.), Elsevier, New York, 1981, attempted to synthesize relaxin using the separate chain approach using selective protection of the cysteine sulfhydryls. They found that the synthetic peptides prepared by the insulin methods had detectable relaxin-like bioreactivity however, the specific activity and combination yields were very low. Id. at 151. One reason for the poor combination yields for porcine relaxin was due to the insolubility of the full length procine B-chain in solution at pH 10.5 Tregear et al., supra. modified the prior insulin combination methodology in two respects: by precipitation of the mixed porcine relaxin peptide chains with acetone to remove the reducing agent; and by adding 0.5M NaCl during the oxidation step. The results were improved yields of porcine relaxin. Also see European Patent Application No. 83.304662.6 and European Patent Application No. 83307553.4, EP Pub. Nos. 101,309 and 112,149, respectively.
Chance et al., U.S. Pat. No. 4,421,685 issued Dec. 20, 1983 disclose a method for producing insulin or an analog thereof by combining the S-sulfonated form of the insulin A- and B-chains with a thiol reducing agent in an aqueous medium under controlled pH and temperature so as to carry out the reduction and oxidation reactions in a single step. This method, while presented as an improvement over the previously mentioned insulin synthesis, was found not to be applicable to the synthesis of human relaxin.
It has now been discovered that under specific reaction conditions useful levels of human relaxin or analogs thereof can be produced by combining the reduced chains of human relaxin under controlled conditions. Thus it is an object of the present invention to provide a method for combining the A- and B-chains of human relaxin, regardless of their origin, e.g. chemical synthesis or recombinant DNA technology, to produce useful yields of biologically active human relaxin.
Another aspect of the invention is to produce biologically active analogs of human relaxin.
Yet another aspect of the invention is the use of human relaxin or an analog thereof to effect parturition.